The present invention relates generally to surface plasmon resonance sensors. In particular, the present invention relates to instruments, methods and reagents for amplifying the surface plasmon resonance response and increasing its sensitivity, flexibility, throughput, specificity, and scope.
Surface plasmon resonance (SPR) is a general spectroscopic method for sensing refractive index changes near the surface of a metal film. Its sensitivity to these changes provides a versatile platform for the observation and quantitation of chemical reactions at the metal/solution interface, provided the chemistry is well-designed. The generality of the technique has led to its application to a variety of chemical systems, including biosensing (where specifically designed commercial instrumentation is available).
SPR allows detection of small changes in refractive index that result from interactions between surface-confined biomolecules and solution-borne species. For example, immobilization of a protein to the sensor surface allows for detection of protein binding events manifested by a change in refractive index and hence a change in the angle-dependent reflectance of the metal film. This type of SPR sensing is typically carried out on commercial instruments that use a carboxylated dextran gel on a Au film as the sensor surface, where the gel acts as a host for the surface-confined binding partner. However, SPR has been applied in a number of other formats, including imaging SPR where a large number of chemistries can be rapidly interrogated simultaneously. Likewise, a variety of other surface chemistries have been used as scaffolds for measurements of biomolecular interactions.
SPR relies on the optical excitation of surface modes (plasmons) in a free electron metal (e.g., a 50 nm thick film of Au, Ag, Al, or Cu anchored to a glass substrate by a thin adhesion layer of Ti, Cr, or mercaptosilane). Back-side, p-polarized illumination of a prism-coupled film at some angle greater than the critical angle for total internal reflection results in plasmon excitation at the metal-solution interface. Plasmon excitation is observed as an increase in optical absorbance (decrease in reflectance) at an optimal coupling angle. This, in turn, results in a minimum in the SPR profile (a plot of reflectance versus angle), which is referred to as the plasmon angle (xcex8p). Sensing via SPR is possible due to the sensitivity of xcex8p to changes in the index of refraction near the metal surface. Adsorption, desorption, and molecule-molecule interactions that occur at the metal-solution interface result in such changes, thereby inducing a shift in plasmon angle. These changes can be monitored in real-time, making SPR suitable for dynamic sensing.
Perhaps the most widely studied subset of chemistries using SPR is protein-protein interactions, where binding event signal transduction is difficult or impossible to accomplish by traditional optical spectroscopies. In order to decrease nonspecific binding and to increase surface loading of biomolecules, SPR experiments conducted on commercial instrumentation commonly use an extended coupling matrix in conjunction with the sensing surface. Such measurements typically begin with one protein immobilized on proprietary substrates comprising a carboxylated dextran (or xe2x80x9cextended couplingxe2x80x9d) matrix layered on top of a thin evaporated Au film (i.e., between the film and the sample). The result is an extended three-dimensional array of molecules extending some 200 nm away from the surface of the Au film. Protein binding events leading to small changes in the refractive index of the dextran layer are detected via correspondingly small changes in the angle-dependent attenuated total reflectance. Despite the signal amplification afforded by the dextran matrix, the detection of small ( less than 1000 MW) molecules is still a non-trivial task for commercial instrumentation. In some cases even the detection of species in the 2,000-10,000 MW range can prove challenging.
Use of coupling matrices is associated with a number of additional drawbacks, including the possibility of nonspecific interactions with biomolecules that dominate the signal, and the exclusion of large proteins. Furthermore, improper orientation of biomolecules within the matrix often leads to low biomolecular activity, especially with proteins. Because mass transport to molecules immobilized in the matrix is commonly pH dependent, separate steps are required to optimize diffusion of reagents into the matrix during the assay. These shortcomings can create significant difficulties in assay design. It would be desirable to avoid these problems by using planar SPR substrates (i.e., without an overlying matrix) such as Au films modified with a monolayer of a bifunctional organic crosslinker. However, SPR reflectivity changes within these more uniform substrates are often too small to be measurable in any practical assay.
The applicability and usefulness of SPR could be greatly expanded if ligand binding events resulted in more pronounced changes in refractive index and, hence, more pronounced shifts in plasmon angle. Such increased sensitivity could make the technique broadly applicable to high-throughput screening of low molecular-weight drug candidates.
It is an object of the present invention to provide instruments, methods and reagents for the amplification of SPR reflectivity changes in chemical assays, especially in biomolecular recognition assays on planar SPR substrates coated with a monolayer or submonolayer of capture reagents (e.g., antibodies). It is also an object of the invention to provide methods and reagents for ultra-sensitive, non-PCR-based DNA detection assays. It is moreover an object of this invention to describe an SPR-based method for measuring distances between surface-confined objects on the scale from 1 to 500 nm, with 3-Angstrom resolution.
An additional object of this invention is to enable high throughput screening reactions (both primary and secondary) by amplified imaging SPR.
A further object of the invention is to provide a xe2x80x9cwet chemistryxe2x80x9d method for the synthesis of Au films for SPR (or other surface spectroscopies) as a replacement for (or alternative to) cumbersome evaporative methods used in the art.
Finally, it is an object of the invention to provide an imaging SPR instrument capable of depicting spatial differences in film reflectance at fixed angles of incidence, which spatial differences are induced by differential indices of refraction or film thickness (i.e., differential chemical modification). The imaging SPR instrument provided by the invention allows SPR to be used in multiplexed biological xe2x80x9cchipxe2x80x9d assay formats. For example, the imaging SPR instrument would be integral in the simultaneous detection of multiple target analytes using a solid support to which ligands for the different target analytes are attached at specific locations.
The instrumentation, methods, and reagents of the present invention enable chemical assays (including multiplexed biosensing assays) of unprecedented sensitivity and selectivity.
The present invention provides instrumentation, methods, and reagents for the amplification of SPR reflectivity changes. In one series of embodiments, colloidal-metal nanoparticles are used as optical tags for SPR-based sensing assays. These embodiments rely on the observation that the SPR response of a metal film changes dramatically upon localization of such colloidal-metal nanoparticles to the film surface. The dramatic change in the SPR response of metal films that occurs upon adsorption of colloidal metal nanoparticles can be exploited in any assay that depends on the occurrence of a molecular recognition event (e.g., the binding of antigen to an antibody, the binding of a ligand to its receptor, or the hybridization of complementary nucleic acid molecules). In one of the many possible implementations, one of the molecules that participates in a molecular recognition event is immobilized on the surface of a metal film of the SPR substrate (or to an entity attached to the metal film). Another molecule that participates in the interaction with the immobilized moleculexe2x80x94either by binding directly to the immobilized molecule, or by binding to a third molecule that in turn binds to the immobilized moleculexe2x80x94is then tagged with the colloidal metal nanoparticle. The binding between the participating molecules leads to colloidal metal adsorption, with the concomitant change in the SPR response of the film. Methods are well known in the art for preparing monodisperse colloidal metal nanoparticles in solution, as are methods for attaching biomolecules to the metal nanoparticles without loss of biological activity. In some embodiments, the use of colloidal Au nanoparticles leads to a 100,000-fold increase in SPR sensitivity.
For example, colloidal Au nanoparticles can be readily attached to ligands of target analytes. Then, when the target analyte becomes localized to the surface of the SPR film (or to ligands immobilized thereon), the Au-tagged ligands are brought to the surface when the ligands bind the target analyte, leading to the amplified changes in SPR reflectivity. The amplification in signal is sufficiently large that planar SPR substrates (such as a Au film modified with a bifunctional organic cross linker) can be used, thus avoiding several of the problems associated with commercial dextran-based matrices.
Notwithstanding the significant limitations discussed above, dextran-based SPR substrates are adequate for some types of assay. For this reason, the invention also provides methods for the amplification of SPR reflectivity within dextran-based matrices. In some embodiments of the invention, Au nanoparticles attached to ligands are first brought to the matrix and retained there through the biomolecular interaction that forms the basis of the assay. The nanoparticles are chosen to be of a sufficiently small diameter to permit efficient diffusion into the network. However, nanoparticles of this size do not optimally amplify changes in SPR reflectivity. Consequently, to optimize amplification, the nanoparticles are then enlarged through addition of an Ag plating solution.
In other embodiments, the invention provides a method for further enhancing colloidal Au-amplified SPR reflectivity by overlaying an inorganic film over the Au surface. The distance between the metallic nanoparticles and the metal surface has a significant impact on SPR reflectivity. In preferred embodiments, a 30 nm thick film of SiO2 is evaporated onto a 50 nm Au surface. When colloidal gold nanoparticles become attached to this surfacexe2x80x94either directly or by way of, e.g., a protein-protein interactionxe2x80x94the SiO2 layer acts as a spacer to optimize the coupling distance between the Au nanoparticles and the Au surface. This leads to a greater shift in the plasmon angle (i.e., angle of minimum reflectance) than observed using colloidal Au nanoparticles and a Au surface without an SiO2 film. The enhancement is observed even when the Au surface is bound to very low levels of colloidal Au (e.g., less than 1% surface coverage).
In another series of embodiments, the methods are used to provide a xe2x80x9csandwichxe2x80x9d immunoassay. Specifically, a primary xe2x80x9ccapturexe2x80x9d antibody to a target analyte is immobilized on the surface of an SPR substrate, either on the Au surface itself, or on an overlaying SiO2 film (or other material). A secondary antibody to the target analyte is conjugated to colloidal Au nanoparticles by one of a variety of methods. The Au nanoparticles and the SPR substrate are then contacted with a biological fluid suspected of containing the target analyte. If present, the target analyte will become bound to the SPR substrate through its interaction with the immobilized capture antibody, and the colloidal Au will become localized to the SPR substrate through the interaction between the secondary antibody and the bound target analyte. As a result, an amplified change in the plasmon angle will be observed, amplified with respect to the angle shift observed if the secondary antibody was not conjugated to colloidal Au nanoparticle. In some cases, the methods provided herein provide a 100,000-fold or greater enhancement in SPR signal relative to conventional SPR assays using dextran-based capture matrices, and provide the capability for detecting protein concentrations of less than 70 fM. It should be clear to those skilled in the art that such assays can be carried out in separate steps, in which the analyte-containing solution is first exposed to the surface, and wash steps are carried out to eliminate non-specific binding. Then, in a second step, the colloidal Au:antibody conjugate is introduced.
In another series of embodiments, the methods of the invention are applied to detect target nucleic acid sequences. For example, a xe2x80x9cfirst nucleic acid probexe2x80x9d may be immobilized on the surface of a planar SPR substrate and a xe2x80x9csecond nucleic acid probexe2x80x9d conjugated to colloidal Au nanoparticles. The probes have regions at least partially complementary to the target nucleic acid sequence (or are capable of some other interaction) such that in the presence of the target sequence, both the probes become associated therewith. As a result, the colloidal Au nanoparticles conjugated to the second nucleic acid probe become localized to the SPR substrate and there is an amplified plasmon angle shift. The methods of the invention enable a quantitation limit of better than 8xc3x97107 molecules/cm2 for a 24-mer oligonucleotide with approximately 5 decades of dynamic range. This is more than a 1000-fold improvement in the sensitivity observed without using the amplifying methods of the invention. In this embodiment as well, introduction of the second nucleic acid probe can occur before, at the same time, or after introduction of the target sequence.
The invention also provides an SPR instrument capable of operating in imaging mode rather than scanning mode. Scanning mode SPR measures a single SPR reflectivity value for the entire SPR substrate at each angle of incidence (and/or wavelength of the incident light) within a specified range. The imaging mode instrument uses a charge-coupled device (CCD) to provide an image that depicts local SPR reflectivity values throughout an SPR substrate at one or more predetermined angles of incidence. The angle(s) of incidence chosen is the angle at which the largest change in SPR reflectivity is observed upon occurrence of the event to be assayed (e.g., upon the binding of an immobilized ligand to its target). This enables SPR to be used as the basis for detection in microarray format for the purpose of multiplexed assays. In such assays, the SPR instrument captures a normalized reflectance image of the SPR substrate after addition of the fluid suspected of containing the target analyte. In the resulting image, the intensity of the reflectance signal in each xe2x80x9cspotxe2x80x9d on the substrate yields information about the amount of target analyte present at that location. The imaging SPR apparatus described herein allows for the simple introduction of large samples (up to 50 mmxc3x9750 mm microarray substrates, using commercially available optics). Moreover, with custom optics, much larger surfaces, e.g., 6 inchesxc3x976 inches, can be used. Unlike previously described SPR instruments, the imaging device of the present invention provides a unique horizontal sample geometry that enables the robotic manipulators to control substrate handling and liquid delivery. This geometry obviates the need to flow solutions onto a vertical SPR substrate, as is required in prior art instrumentation.
Modifications to particles and/or surfaces aimed at increasing sensitivity and selectivity of SPR include the use of: (i) new types of metal nanoparticles such as AuS/Au core-shell particles, designed to optimize the wavelength-dependent component of dipolar coupling to surfaces; (ii) particles with sophisticated surface functionalization that exhibit reduced non-specific binding and that allow control over the number of biomolecules bound to the particle; (iii) substrates with sub-wavelength holes designed to provide local field enhancement; and (iv) oxide-coated metal films that optimize the distance dependent component of particle-surface coupling. Instrumental approaches to increased sensitivity include the use of tuned excitation wavelengths, measurement of angle-dependent surface plasmon scattering in parallel with SPR, and interferometric SPR. A third line of embodiments to improve SPR sensitivity involves the use of two secondary signal amplification schemes. One is based on particle xe2x80x9cdevelopmentxe2x80x9d, in which selective chemistries are used to grow the dimensions of immobilized particles; a second is based on a cascade effect in which molecular recognition-induced binding of one metal nanoparticle leads to selective deposition of several others from solution.
To define regions of the SPR substrate for different chemistries, a method has been developed for producing disposable replicas of printed circuit boards. The resulting xe2x80x9cnanobeakersxe2x80x9d (fabricated from polydimethylsiloxane, or PDMS) allow the ability to individually address a large number of spatially distinct regions over a small surface. Use of such microwell assays have illustrated that changes in Au particle coverage of less than 0.1% of a monolayer are easily detected by imaging SPR.